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      Fragment Screening at Adenosine-A 3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay

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          Summary

          G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A 1 and -A 3 receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (K D). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A 3 receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.

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          Highlights

          ► Fluorescence-based competition binding assay for adenosine-A 1 and -A 3 receptors ► Fragment screen using receptors in the native membrane environment in living cells ► Lead compound identification and optimization from a commercial fragment library

          Abstract

          G protein-coupled receptors are important drug targets, and initiatives that measure ligand affinity in the native cellular environment are required. Here, Stoddart et al. report a live cell, high content, fluorescence-based assay to measure ligand affinity at the human adenosine-A1 and -A3 receptors.

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          Most cited references39

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          High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.

          Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.
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            International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors--an update.

            In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.
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              Ligand efficiency: a useful metric for lead selection.

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                Author and article information

                Journal
                Chem Biol
                Chem. Biol
                Chemistry & Biology
                Elsevier
                1074-5521
                1879-1301
                21 September 2012
                21 September 2012
                : 19
                : 9
                : 1105-1115
                Affiliations
                [1 ]Institute of Cell Signalling, School of Biomedical Science, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
                [2 ]School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK
                Author notes
                []Corresponding author barrie.kellam@ 123456nottingham.ac.uk
                [∗∗ ]Corresponding author stephen.hill@ 123456nottingham.ac.uk
                Article
                CHBIOL2166
                10.1016/j.chembiol.2012.07.014
                3456874
                22999879
                073c9ed0-325f-4c2a-8ab6-39d536b56a59
                © 2012 Elsevier Ltd.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 26 March 2012
                : 22 June 2012
                : 17 July 2012
                Categories
                Article

                Biochemistry
                Biochemistry

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